Apolipoprotein(a) Gene Enhancer Resides within a LINE Element

Apolipoprotein(a), (apo(a)), is the distinguishing protein portion of the lipoprotein(a) particle, elevated plasma levels of which are a major risk factor for cardiovascular disease. A search for enhancer elements that control the transcription of the apo(a) gene led to the identification of an upstream element that contains target binding sites for members of the Ets and Sp1 nuclear protein families. The enhancer element functions in either orientation to confer a greater than 10-fold increase in the activity of the apo(a) minimal promoter in cultured hepatocyte cells. Unexpectedly, the enhancer element is located within a LINE retrotransposon element, suggesting that LINE elements may function as mobile regulatory elements to control the expression of nearby genes. Apolipoprotein(a), (apo(a)), is the distinguishing protein portion of the lipoprotein(a) particle, elevated plasma levels of which are a major risk factor for cardiovascular disease. A search for enhancer elements that control the transcription of the apo(a) gene led to the identification of an upstream element that contains target binding sites for members of the Ets and Sp1 nuclear protein families. The enhancer element functions in either orientation to confer a greater than 10-fold increase in the activity of the apo(a) minimal promoter in cultured hepatocyte cells. Unexpectedly, the enhancer element is located within a LINE retrotransposon element, suggesting that LINE elements may function as mobile regulatory elements to control the expression of nearby genes. Elevated levels of lipoprotein(a) (Lp(a)) 1The abbreviations used are: Lp(a), lipoprotein(a); apo(a), apolipoprotein(a); HNF, hepatocyte nuclear factor; kb, kilobase pair(s); bp, base pair(s); PCR, polymerase chain reaction; ACR, apo(a) gene transcription control region; L1, LINE element 1; BAC, bacterial artificial chromosome. 1The abbreviations used are: Lp(a), lipoprotein(a); apo(a), apolipoprotein(a); HNF, hepatocyte nuclear factor; kb, kilobase pair(s); bp, base pair(s); PCR, polymerase chain reaction; ACR, apo(a) gene transcription control region; L1, LINE element 1; BAC, bacterial artificial chromosome. are a major risk factor for atherosclerosis, myocardial infarction, and cerebrovascular stroke (see Refs. 1Bostom A.G. Cupples L.A. Jenner J.L. Ordovas J.M. Seman L.L. Wilson P.W.F. J. A. M. A. 1996; 276: 544-548Crossref PubMed Google Scholar and 2Assmann G. Schulte H. von Eckardstein A. Am. J. 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Numerous studies have found a large variation in apo(a) mRNA quantity in humans and primates, which corresponds with plasma concentration in most cases (23Hixson J.E. Britten M.L. Manis G.S. Rainwater D.L. J. Biol. Chem. 1989; 264: 6013-6016Abstract Full Text PDF PubMed Google Scholar, 24Koschinsky M.L. Beisiegel U. Henne-Bruns D. Eaton D.L. Lawn R.M. Biochemistry. 1990; 29: 640-644Crossref PubMed Scopus (121) Google Scholar, 25Wade D.P. Knight B.L. Harders-Spengel K. Soutar A.K. Atherosclerosis. 1991; 91: 63-72Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 26Azrolan N. Gavish D. Breslow J.L. J. Biol. Chem. 1991; 266: 13866-13872Abstract Full Text PDF PubMed Google Scholar). We have previously shown that the liver-specific expression of the apo(a) gene is partly mediated by the apo(a) proximal promoter, which is located from −98 to +130 relative to its transcription start site and responds to the liver-enriched transcription factor HNF-1α (27Wade D.P. Lindahl G.E. Lawn R.M. J. Biol. Chem. 1994; 269: 19757-19765Abstract Full Text PDF PubMed Google Scholar). However, there are several indications that additional transcriptional regulatory elements must contribute to the high levels of apo(a) gene expression: activity of this core promoter is relatively weak in in vitro transfection assays, it does not account for the sex hormone responsiveness of the apo(a) gene, and it contains far less sequence polymorphism than could account for the inherited variation in gene expression, which is known to map to this locus. A further indication that regulatory sequences exist further 5′ to the apo(a) gene comes from transgenic mouse experiments. Deletion of the region 5′ to the apo(a) promoter from a yeast artificial chromosome severely compromises apo(a) expression in transgenic mice compared with a yeast artificial chromosome containing approximately 80 kb of upstream sequence. 2S. Hughes and E. Rubin, personal communication. 2S. Hughes and E. Rubin, personal communication. The apo(a) gene is part of a cluster of genes and pseudogenes on chromosome six which share over 70% sequence identity (28Magnaghi P. Citterio E. Malgaretti N. Acquati F. Ottolenghi S. Taramelli R. Hum. Mol. Genet. 1994; 3: 437-442Crossref PubMed Scopus (43) Google Scholar, 29Byrne C.D. Schwartz K. Meer K.J. Cheng J.F. Lawn R.M. Arterioscler. Thromb. 1994; 14: 534-541Crossref PubMed Google Scholar). The apo(a) and the plasminogen genes are organized in a head to head configuration separated by about 40 kb. We have systematically tested this entire intergene region for additional apo(a) regulatory elements. In this study, we describe an apo(a) transcription control region (ACR) that is located 20 kb 5′ to the apo(a) transcription start site. The enhancer region contains an essential element responding to the members of the Ets transcription factor family. In addition, the binding of Sp1 as well as several other transcription factors around the Ets site also contributes to maximal enhancer activity. Unexpectedly, the apo(a) enhancer region resides within a LINE element. LINE 1 (L1) elements are long interspersed repeat elements that number from 50,000 to 100,000 in the human genome. Although greater than 90% of them are truncated, full-length L1s are approximately 6 kb in length and contain a 5′-untranslated region with internal transcription regulatory elements, two open reading frames, and a 3′-untranslated region that terminates in a poly(A) tail (30Swergold G.D. Mol. Cell. Biol. 1990; 10: 6718-6729Crossref PubMed Scopus (339) Google Scholar, 31Singer M.F. Krek V. McMillan J.P. Swergold G.D. Thayer R.E. Gene (Amst.). 1993; 135: 183-188Crossref PubMed Scopus (53) Google Scholar, 32Scott A.F. Schmeckpeper B.J. Abdelrazik M. Comey C. O'Hara B. Rossiter J. Cooley T. Heath P. Smith K.D. Margolet L. Genomics. 1987; 1: 113-125Crossref PubMed Scopus (263) Google Scholar). is the of an element the expression of a In the from other human elements found to have enhancer activity as well as one more in the core Ets binding site sequence. the element upstream of the apo(a) gene may a of repeat elements that have the to the expression of genes. from (27Wade D.P. Lindahl G.E. Lawn R.M. J. Biol. Chem. 1994; 269: 19757-19765Abstract Full Text PDF PubMed Google with to a unique site and to contains human apo(a) 5′ sequences from an site kb upstream of the transcription start site to an site of the start to the gene in is a similar containing a of the sequence used contains the minimal promoter of apo(a) from −98 to +130 (27Wade D.P. Lindahl G.E. Lawn R.M. J. Biol. Chem. 1994; 269: 19757-19765Abstract Full Text PDF PubMed Google and from by of an and of the site. A human in the with the of apo(a) and plasminogen genes. of the two containing an of kb, to have the entire region as well as of the of apo(a) and plasminogen genes by with apo(a) and plasminogen of as in either of The also with and for the of of the that there is one site in the entire with and In most the tested in one orientation relative to the transcription of on the of and in with in cell to transfection by a to the as we previously (27Wade D.P. Lindahl G.E. Lawn R.M. J. Biol. Chem. 1994; 269: 19757-19765Abstract Full Text PDF PubMed Google Scholar). in of expression and 1 of the control control with 1 and of expression to well with of the and and and the to the control for transfection efficiency, are as the of to activity. of in and 6 the of a site from the upstream of and the one located of L1, to the of Swergold (30Swergold G.D. Mol. Cell. Biol. 1990; 10: 6718-6729Crossref PubMed Scopus (339) Google from either the the orientation of the and in and are the internal of from and by with and either the the orientation of the and by a of site in in with and to the region to of to the with and as the The internal the in A. The and and by to transfection the enhancer containing with a promoter, it and sites of the upstream of the from as previously (27Wade D.P. Lindahl G.E. Lawn R.M. J. Biol. Chem. 1994; 269: 19757-19765Abstract Full Text PDF PubMed Google Scholar). and previously D. H. M. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar, J.L. 1989; 3: PubMed Scopus Google Scholar). The of site used for is The containing and used for the the as site A the Ets site as in A. The from and and plus and from the of by the site with and the by The sequence of the Sp1 used in the is The locus of the apo(a) and plasminogen genes is shown in The apo(a) and plasminogen genes are organized in a head to head configuration with about a region them region in a which contains a the region plus of the apo(a) and the plasminogen genes and the intergene region in of the apo(a) minimal promoter and We previously the core promoter region that −98 to +130 relative to the transcription start site of the human apo(a) gene and responds to the liver-enriched transcription factor HNF-1α (27Wade D.P. Lindahl G.E. Lawn R.M. J. Biol. Chem. 1994; 269: 19757-19765Abstract Full Text PDF PubMed Google Scholar). two activity the containing the core apo(a) of contains a previously plasminogen enhancer located kb upstream of the plasminogen transcription start site G. R. Taramelli R. J. 1996; PubMed Scopus Google Scholar). is not to that it transcription in vitro from apo(a) and plasminogen core the two promoter contain about 90% sequence The of the plasminogen enhancer on apo(a) gene expression to The most of apo(a) transcription a that is located about 20 kb upstream of the apo(a) promoter 1 and with one of the four liver-specific by P. A. Taramelli R. 1994; PubMed Scopus Google Scholar). is of apo(a) transcription in an and in the enhancer elements in this it to shown in of the 5′ of the relative to the apo(a) in a of the enhancer the of essential elements within this By this is to confer an over 10-fold activation A further a activity. We that the enhancer domain resides within the which a greater than 10-fold activation when in either orientation 5′ to the apo(a) core promoter and and We this the apo(a) transcription control region of the of the and in an increase of activity compared with the entire due either to elements in to the in activity by the size of the transcription of the apo(a) gene is by the core promoter region and not the We previously shown that the apo(a) core promoter can transcription in not in (27Wade D.P. Lindahl G.E. Lawn R.M. J. Biol. Chem. 1994; 269: 19757-19765Abstract Full Text PDF PubMed Google Scholar). When to the core promoter, the is to transcription in activity in cells. In when to the promoter, it can transcription in cell not sequence that the is located in the 5′-untranslated region of a highly element. The is located from relative to the transcription start site of the element by Swergold (30Swergold G.D. Mol. Cell. Biol. 1990; 10: 6718-6729Crossref PubMed Scopus (339) Google Scholar). the the enhancer activity is to is a of the this and to used to by this region from human elements. When the expression of the from 20 to of the activity shown by the in not of the several with the sequence human elements. two sites A and found to between the and the The is the one that contains a in its sequence within site of the L1s either this site the sequence of the is a in not site shown to the may a of element that can as a more enhancer the apo(a) gene when located in its The sequence containing the site unique to the ACR, resembles the binding site for the Ets of transcription factors J.M. B.J. 1992; 6: PubMed Scopus Google Scholar). to its in expression from the apo(a) core shown in and the activity of the The of as factors by of the with an expression for the The expression expression in a However, protein to the expression to the when the Ets binding site is The activity by with in this may the of other weak Ets binding sites in the of the on the activity of the core promoter region in that members of the Ets transcription factor are of apo(a) gene expression through site A in the of the of this site by site A used as a to with nuclear from A that by a of less by site A and and an with the of an factor binding site in the apo(a) of the to other binding sites that may contribute to the enhancer activity. from of the region from with of the sequence to the binding sites of transcription factors in most of the Sp1 and with site the previously Ets binding site. A of Ets binding to the major of is a region in the and a site on the similar to the in J.M. B.J. 1992; 6: PubMed Scopus Google Scholar). The of and by a When that the region Sp1 it the Sp1 binding Refs. M. J. S. R. Science. PubMed Scopus Google Scholar and J. R. Cell. 1987; Full Text PDF PubMed Scopus Google with the and nuclear from and the of sites by in and in to the Ets protein, transcription factor Sp1 is in binding to the The identity of other binding in this region the effects of the other binding sites within the ACR, we the entire to further and in sites and tested in transfection in cells. A of the sites and from with shown in for base as a a shown in of and as well as of the 50 which of and most of not cause a significant in expression, compared with of the Deletion of the region and also significant not is that the in site which to one of the two sites the sequence of the from the In of the binding sites for and reduce the enhancer activity. The identity of the transcription factors which to and are the target site for a of the Ets is a likely target of an We the of the 40 kb that between the 5′ of the human apo(a) and plasminogen genes for elements other than the apo(a) core promoter that the expression of the apo(a) gene in an in vitro We have an apo(a) transcriptional control region located between the apo(a) and the plasminogen that is to confer a greater than 10-fold activation when linked in either orientation to the apo(a) minimal of the it to part of a LINE repeat element, members number to 100,000 in the human genome. of of other human elements that they are less than the in the activity of the apo(a) core promoter, and that they contain sequence in a site to the target of the Ets of transcription We also additional sequences in and found that they from the in the Ets target sequence. this Ets binding site to for the apo(a) enhancer activity. of an expression a activation of expression, further the of and further the of additional factors Sp1 in the transcription enhancer activity and likely between several binding in the from have shown that the apo(a) minimal promoter is mediated by a liver-enriched transcription factor HNF-1α and can confer expression (27Wade D.P. Lindahl G.E. Lawn R.M. J. Biol. Chem. 1994; 269: 19757-19765Abstract Full Text PDF PubMed Google Scholar). In and more enhancer elements control the expression of genes. In the of apo(a), the does not to suggesting that the apo(a) proximal promoter is to of its the of significant apo(a) levels of its mRNA have also in and in of J.E. J.W. Lawn R.M. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google human isoform of apo(a) from to H.G. Menzel H.J. F. Utermann G. J. Clin. Invest. 1989; PubMed Scopus Google The may function by the expression of the apo(a) of an between liver-specific and elements of an is found in the gene, is by a control region that is located kb from the start of the gene N. J.M. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). A number of other genes are known to by transcription elements located of from transcription start sites N. J.M. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, A. Azrolan N. K. A. A. Breslow J.L. 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